Certain products and byproducts of naturally occurring metabolic processes in cells can be used for a wide spectrum of industries, including the animal feed industry, food industry, cosmetics industry and pharmaceuticals industry. These molecules, which are joinly referred to as “fine chemicals”, also include lipids and fatty acids, amongst which the polyunsaturated fatty acids constitute an example of one class. Polyunsaturated fatty acids (PUFAs) are added, for example, to children's formula to increase its nutritional value. For example, PUFAs have a positive effect on the cholesterol level in the blood of humans and are therefore suitable for protection against heart disease. Fine chemicals and polyunsaturated fatty acids (PUFAs) can be isolated from animal sources, for example fish, or microorganisms. Culturing these microorganisms allows large amounts of one or more of the desired molecules to be produced and isolated.
Microorganisms which are especially suitable for preparing PUFAs are microorganisms such as Thraustochytria or Schizochytria strains, algae such as Phaeodactylum tricornutum or Crypthecodinium species, Ciliata such as Stylonychia or Colpidium, fungi such as Mortierella, Entomophthora or Mucor. A number of mutant strains of the microorganisms in question which produce a series of desirable compounds, including PUFAs, have been developed by strain selection. The selection of strains with an improved production of a certain molecule is, however, a time consuming and difficult procedure. Also disadvantageous is the fact that only specific unsaturated fatty acids, or only a specific fatty acid spectrum, can be produced by a defined microorganism.
As an alternative, fine chemicals can suitably be produced on a large scale via the production of plants which have been developed in such a way that they produce the abovementioned PUFAs. Plants which are particularly well suited to this purpose are oil crops which contain large amounts of lipid compounds, such as oilseed rape, canola, linseed, soya, sunflowers, borage and evening primrose. However, other crops which contain oils or lipids and fatty acids are well suited, as mentioned in the detailed description of the present invention. Conventional plant breeding has led to the development of a series of mutant plants which produce a spectrum of desirable lipids and fatty acids, cofactors and enzymes. However, the selection of novel plant varieties with an improved production of a certain molecule is a time-consuming and difficult procedure or even impossible if the compound does not occur naturally in the plant in question, such as in the case of polyunsaturated C20-fatty acids, and C22-fatty acids and those with longer carbon chains.
The invention provides novel nucleic acid molecules which are suitable for identifying and isolating elongase genes of PUFA biosynthesis and which can be used for the modification of oils, fatty acids, lipids, lipid-derived compounds and, most preferably, for the preparation of polyunsaturated fatty acids, since there remains a great demand for novel genes which encode enzymes which are involved in the biosynthesis of unsaturated fatty acids and which make it possible for these to be prepared on an industrial scale. In particular, there is a demand for fatty acid biosynthesis enzymes which make possible the elongation of polyunsaturated fatty acids, preferably with two or more double bonds in the molecule. The nucleic acids according to the invention encode enzymes which have this ability.
Microorganisms such as Phaeodactylum, Colpidium, Mortierella, Entomophthora, Mucor, Crypthecodinium and other algae and fungi and plants, in particular oil crops, are generally used in industry for the production of a large number of fine chemicals on a large scale.
As long as cloning vectors and techniques are available for the genetic manipulation of the abovementioned microorganisms and Ciliata, as disclosed in WO 98/01572 and WO 00/23604, or algae and related organisms, such as Phaeodactylum tricornutum, described in Falciatore et al. [1999, Marine Biotechnology 1(3):239-251]; and Dunahay et al. [1995, Genetic transformation of diatoms, J. Phycol. 31:10004-1012] and the references cited therein, the nucleic acid molecules according to the invention can be used for the recombinant modification of these organisms so that they become better or more efficient producers of one or more fine chemicals, especially unsaturated fatty acids. This improved production or production efficiency of a fine chemical can be caused by a direct effect of manipulating a gene according to the invention or by an indirect effect of this manipulation.
Mosses and algae are the only known plant systems which produce considerable amounts of polyunsaturated fatty acids, such as arachidonic acid (=ARA) and/or eicosapentaenoic acid (=EPA) and/or docosahexaenoic acid (=DHA). Mosses contain PUFAs in membrane lipids, while algae, organisms related to algae and some fungi also accumulate considerable amounts of PUFAs in the triacylglycerol fraction. Thus, nucleic acid molecules which are isolated from such strains which also accumulate PUFAs in the triacylglycerol fractions are particularly suitable for modifying the lipid and PUFA production systems in a host, in particular in microorganisms, such as the abovementioned microorganisms, and plants, such as oil crops, for example oilseed rape, canola, linseed, soya, sunflower, borage, castor-oil plant, oil palm, safflower (Carthamus tinctorius), coconut, peanut or cacao bean. Furthermore, nucleic acids from triacylglycerol-accumulating microorganisms can be used for identifying such DNA sequences and enzymes in other species which are suitable for modifying the biosynthesis of PUFA precursor molecules in the organisms in question. Microorganisms which accumulate PUFAs such as ARA, EPA or DHA in triacylglycerols are, in particular, microorganisms such as Crypthecodinium cohnii and Thraustochytrium species. Thraustochytria are also closely related to the Schizochytria strains in terms of phylogenetics. Even though these organisms are not closely related to mosses such as Physcomitrella, sequence similarities at the DNA sequence and, in particular, polypeptide level can be observed to such an extent that DNA molecules can be identified, isolated and characterized functionally in heterologous hybridization experiments, sequence alignments and experiments using the polymerase chain reaction, even from organisms which are distantly related in terms of evolution. In particular, consense sequences can be derived which are suitable for the heterologous screening or the functional complementation and prediction of gene functions in third species. The ability to identify these functions, for example to predict the substrate specificity of enzymes, can therefore be of significant importance. Furthermore, these nucleic acid molecules may act as reference sequences for mapping related genomes or for deriving PCR primers.
The novel nucleic acid molecules encode proteins termed in the present context PUFA-specific elongases (=PSEs, or PSE in the singular). These PSEs can, for example, exert a function which is involved in the metabolism (for example in the biosynthesis or in the breakdown) of compounds required for lipid or fatty acid synthesis, such as PUFAs, or which participate in the transmembrane transport of one or more lipid/fatty acid compositions, either into the cell or out of the cell.
This novel application shows the isolation of such novel elongase genes in greater detail. For the first time, we have isolated elongase genes which are suitable for producing long-chain polyunsaturated fatty acids, preferably having more than eighteen or twenty carbon atoms in the carbon skeleton of the fatty acid and/or at least two double bonds in the carbon chain while being derived from typical organisms which contain high amounts of PUFAs in the triacylglycerol fraction. This means, in the singular, a PSE gene or PSE protein or, in the plural, PSE genes or PSE proteins. Other known patent applications and publications disclose, or show, no functionally active PSE gene, even though various known patent applications exist which show the elongation of saturated fatty acids of short or medium chain length (WO 98/46776 and U.S. Pat. No. 5,475,099) or the elongation or production of long-chain fatty acids, but which then have no more than one double bond or lead to long-chain fatty acid wax esters (see WO 98/54954, WO 96/13582, WO 95/15387). The invention presented here describes the isolation of novel elongases with novel properties. Starting from the sequence stated in SEQ ID NO:1, it was possible to find further nucleic acids which encode elongases which elongate unsaturated fatty acids.
WO 99/64616, WO 98/46763, WO 98/46764 and WO 98/46765 describe the production of PUFAs in transgenic plants and demonstrate the cloning and functional expression of corresponding desaturase activities, in particular from fungi, but demonstrate no PSE-encoding gene and no functional PSE activity. The expression of the desaturase activities leads to a shift in the fatty acid spectrum in the transgenic plants, but no increased content of unsaturated fatty acids was observed. The production of a trienoic acid with C18-carbon chain has been demonstrated and claimed with reference to gamma-linolenic acid, but the production of very long-chain polyunsaturated fatty acids (with a C20— and longer carbon chain and of trienoic acids and higher unsaturated types) has, however, not been demonstrated to date.
To prepare long-chain PUFAs, the polyunsaturated C16- or C18-fatty acids must be elongated by at least two carbon atoms by the enzymatic activity of an elongase. The nucleic acid sequence SEQ ID NO:1 according to the invention enclodes the first plant elongase which is capable of elongating the C16- or C18-fatty acids with at least two double bonds in the fatty acid by at least two carbon atoms. After one elongation cycle, this enzyme activity leads to C20-fatty acids, and after two, three and four elongation cycles to C22-, C24- or C26-fatty acids. Longer-chain PUFAs can also be synthesized with the aid of the other elongases which are disclosed (SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11). They can be employed individually, multiply or, for example, in addition to the PUFA elongase from the moss Physcomitrella patens (SEQ ID NO:1) for increasing the PUFA content in a novel process for the preparation of PUFAs. The activity of the elongases according to the invention preferably leads to C20-fatty acids with at least two double bonds in the fatty acid molecule, preferably with three or four double bonds, especially preferably three double bonds, in the fatty acid molecule and/or C22-fatty acids with at least two double bonds in the fatty acid molecule, preferably with four, five or six double bonds, especially preferably with five or six double bonds, in the molecule. After the elongation by the enzyme according to the invention has taken place, further desaturation steps may be carried out in order to obtain the highly desaturated fatty acids. The products of the elongase activities and of the further desaturation with is a possibility therefore lead to preferred PUFAs with a higher degree of desaturation, such as docosadienoic acid, arachidonic acid, ω6-eicosatrienedihomo-γ-linolenic acid, eicosapentaenoic acid, ω3-eicosatrienoic acid, ω3-eicosatetraenoic acid, docosapentaenoic acid or docosahexaenoic acid. Substrates of the enzyme activity according to the invention are, for example, taxol acid; 7,10,13-hexadecatrienoic acid, 6,9-octadecadienoic acid, linolic acid, linolenic acid, α- or γ-linolenic acid or stearidonic acid, and also arachidonic acid, eicosatetraenoic acid, docosapentaenoic acid, eicosapentaenoic acid. Preferred substrates are linolic acid, γ-linolenic acid and/or α-linolenic acid, and also arachidonic acid, eicosatetraenoic acid, docosapentaenoic acid and eicosapentaenoic acid. Arachidonic acid, docosapentaenoic acid and eicosapentaenoic acid are especially preferred. The C16- or C18-fatty acids with at least two double bonds in the fatty acid can be elongated by the enzymatic activity according to the invention in the form of the free fatty acid or in the form of the esters, such as phospholipids, glycolipids, sphingolipids, phosphoglycerides, monoacylglycerol, diacylglycerol or triacylglycerol.
Of particular importance for human nutrition is conjugated linolic acid “CLA”. CLA is to be understood as meaning, in particular, fatty acids such as C18:29 cis, 11trans or the isomer C18:210trans, 12 cis, which can be desaturated or elongated after uptake in the body owing to human enzyme systems and can contribute to health-promoting effects. Elongases according to the invention also allow those conjugated fatty acids which have at least two double bonds in the molecule to be elongated and thus make available such health-promoting fatty acids for human nutrition. Other examples of conjugated fatty acids are alpha-parinaric acid, eleostearic acid and calendulic acid.
Given cloning vectors for use in plants and in the transformation of plants, such as those which are published, and cited, in: Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Fla.), chapter 6/7, pp. 71-119 (1993); F. F. White, Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press, 1993, 15-38; B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press (1993), 128-143; Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991), 205-225)), the nucleic acids according to the invention can be used for the recombinant modification of a broad spectrum of plants so that they become a better, more efficient or modified producer of one or more lipid-derived products, such as PUFAs. This improved production or production efficiency of a lipid-derived product, such as PUFAs, can be caused by the direct effect of the manipulation or by an indirect effect of this manipulation.
There exists a series of mechanisms by which the modification of a PSE protein according to the invention can directly affect yield, production and/or production efficiency of a fine chemical from an oil crop or a microorganism, owing to a modified protein. The number or activity of the PSE protein or PSE gene can be increased so that greater quantities of these compounds are produced de novo since the organisms lacked this activity and biosynthesis ability prior to introduction of the gene in question. Also, the use of various, divergent sequences, i.e. sequences which differ at the DNA sequence level, may be advantageous in this context.
The introduction of a PSE gene or a plurality of PSE genes to an organism or a cell can not only increase the biosynthesis flow toward the end product, but also increase, or create de novo, the corresponding triacylglycerol composition. Equally, the number or activity of other genes which are involved in the import of nutrients required for the biosynthesis of one or more fine chemicals (for example fatty acids, polar and neutral lipids) may be increased, so that the concentration of these precursors, cofactors or intermediates is increased within the cells or within the storage compartment, thus further increasing the ability of the cells to produce PUFAs, as described hereinbelow. Fatty acids and lipids themselves are desirable as fine chemicals; optimization of the activity, or increasing the number, of one or more PSEs which are involved in the biosynthesis of these compounds, or destroying the activity of one or more PSEs which are involved in the breakdown of these compounds, can make possible an increase in yield, production and/or production efficiency of fatty acid molecules and lipid molecules from plants or microorgansims.
The mutagenesis of the PSE gene according to the invention may also lead to a PSE protein with modified activities which directly or indirectly affect the production of one or more desired fine chemicals. For example, the number or activity of the PSE gene according to the invention can be increased, so that the normal metabolic waste products or byproducts of the cell (whose quantity might be increased owing to the overproduction of the desired fine chemical) are exported in an efficient manner before they destroy other molecules or processes within the cell (which would reduce cell viability) or would interfere with the biosynthetic pathways of the fine chemical (thus reducing yield, production or production efficiency of the desired fine chemical). Furthermore, the relatively large intracellular quantities of the desired fine chemical themselves may be toxic to the cell or may interfere with enzyme feedback mechanisms, such as allosteric regulation; for example, they might increase the allocation of the PUFA into the triacylglycerol fraction owing to an increased activity or number of other enzymes or detoxifying enzymes of the PUFA pathway which follow downstream; the viability of the seed cells might increase which, in turn, leads to better development of cells in culture or to seeds which produce the desired fine chemical. Alternatively, the PSE gene according to the invention can be manipulated in such a way that the corresponding quantities of the various lipid molecules and fatty acid molecules are produced. This can have a decisive effect on the lipid composition of the cell membrane and generates novel oils in addition to the occurrence of PUFAs which have been synthesized de novo. Since each type of lipid has different physical properties, a change in the lipid composition of a membrane can substantially modify membrane fluidity. Changes in membrane fluidity can have an effect on the transport of molecules via the membrane and on cell integrity, both of which have a decisive effect on the production of fine chemicals. In plants, moreover, these changes can also have an effect on other traits such as the tolerance to abiotic and biotic stress situations.
Biotic and abiotic stress tolerance is a general trait which it is desirable to impart to a broad spectrum of plants such as maize, wheat, rye, oats, triticale, rice, barley, soybean, peanut, cotton, oilseed rape and canola, cassava, pepper, sunflower and tagetes, Solanaceae plants such as potato, tobacco, aubergine and tomato, Vicia species, pea, alfalfa, shrub plants (coffee, cacao, tea), Salix species, trees (oil palm, coconut) and perennial grasses and fodder crops. As a further embodiment according to the invention, these crops are also preferred target plants for genetic engineering. Very especially preferred plants according to the invention are oil crops such as soybean, peanut, oilseed rape, canola, sunflower, safflower, trees (oil palm, coconut) or crops such as maize, wheat, rye, oats, triticale, rice, barley, alfalfa, or shrub plants (coffee, cacao, tea).
Accordingly, one aspect of the invention relates to isolated nucleic acid molecules (for example cDNAs), encompassing nucleotide sequences which encode a PSE or several PSEs or biologically active parts thereof, or nucleic acid fragments which are suitable as primers or hybridization probes for the detection or amplification of PSE-encoding nucleic acids (for example DNA or mRNA). In a specially preferred embodiment, the nucleic acid molecule encompasses one of the nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11, or the coding region or a complement of one of these nucleotide sequences. In other especially preferred embodiments, the isolated nucleic acid molecule according to the invention encompasses a nucleotide sequence which hybridizes with a nucleotide sequence as shown in the sequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11, or a part thereof or which has at least approximately 50%, preferably at least approximately 60%, more preferably at least approximately 70%, 80% or 90% and even more preferably at least approximately 95%, 96%, 97%, 98%, 99% or more homology thereto. In other preferred embodiments, the isolated nucleic acid molecule encodes one of the amino acid sequences shown in the sequence SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12. Preferably, the preferred PSE gene according to the invention also has at least one of the PSE activities described herein.
In a further embodiment, the isolated nucleic acid molecule encodes a protein or part thereof, the protein or the part thereof comprising an amino acid sequence which has sufficiently homology with an amino acid sequence of the sequence SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12, that the protein or the part thereof retains a PSE activity. Preferably, the protein or the part thereof which is encoded by the nucleic acid molecule retains the ability to participate in the metabolism of compounds required for the synthesis of cell membranes of plants or in the transport of molecules via these membranes. In one embodiment, the protein encoded by the nucleic acid molecule has at least approximately 50%, preferably at least approximately 60% and more preferably at least approximately 70%, 80% or 90% and most preferably at least approximately 95%, 96%, 97%, 98%, 99% or more homology with an amino acid sequence of the sequence SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12. In a further preferred embodiment, the protein is a full-length protein, parts of which are essentially homologous to a complete amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12, (which is due to the open reading frame shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11) and which can be isolated in its full length by methods and experiments with which the skilled worker is familiar.
In another preferred embodiment, the isolated nucleic acid molecule originates from Phytophthora infestans, Physcomitrella patens, Crypthecodinium cohnii or Thraustochytrium and encodes a protein (for example a PSE fusion protein) comprising a biologically active domain which has at least approximately 50% or more homology with an amino acid sequence of the sequence SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12 and retains the ability to participate in the metabolism of compounds required for the synthesis of cell membranes of plants or in the transport of molecules via these membranes or which has at least one of the elongation activities resulting in PUFAs such as ARA, EPA or DHA or their precursor molecules or one of the activities listed in Table 1, and also encompasses heterologous nucleic acid sequences which encode a heterologous polypeptide or regulatory proteins.
TABLE 1Fatty acid profile of five transgenic yeast strains in mol %. The proportions ofγ-linolenic acid which has been added and taken up are emphasized by numbers printedin bold, those of the elongated products are underlined and those of the elongatedγ-linolenic acid are emphasized by numbers printed in bold (last line).Fatty acids[mol %]pYES2pY2PSE1apY2PSE1bpY2PSE1cpY2PSE1d16:017.017.616.416.317.616:1Δ928.026.828.027.925.118:06.56.06.45.66.118:1Δ925.923.527.025.221.418:3Δ6,9,1222.615.713.216.422.820:3Δ8,11,14—10.39.08.67.118:3Δ6,9,12—39.640.534.423.7Elongation
In another embodiment, the isolated nucleic acid molecule is at least 15 nucleotides in length and hybridizes under stringent conditions with a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11. The isolated nucleic acid molecule preferably corresponds to a naturally occurring nucleic acid molecule. More preferably, the isolated nucleic acid molecule encodes naturally occurring Crypthecodinium, Phytophthora or Thraustochytrium PSE or a biologically active part thereof.
A further aspect of the invention relates to vectors, for example recombinant expression vectors, comprising at least one nucleotide molecule according to the invention and host cells into which these vectors have been introduced, in particular microorganisms, plant cells, plant tissues, plant organs or intact plants. In one embodiment, such a host cell can store fine chemicals, in particular PUFAs; to isolate the desired compound, the cells are harvested. The compound (oils, lipids, triacylglycerides, fatty acids) or the PSE can then be isolated from the medium or from the host cell which, in the case of plants, are cells comprising or storing the fine chemicals, most preferably cells of storage tissues such as seed coats, tubers, epidermis cells and seed cells.
Yet another aspect of the invention relates to a genetically modified plant, preferably an oil crop as mentioned above, especially preferably a rapeseed, linseed or Physcomitrella patens plant into which a PSE gene has been introduced. In one embodiment, the genome of oilseed rape, linseed or Physcomitrella patens has been modified by introducing, as transgene, a nucleic acid molecule according to the invention encoding a wild-type or mutated PSE sequence. In another embodiment, an endogenous PSE gene in the genome of the donor organisms Physcomitrella patens, Phytophthora infestans, Crypthecodinium or Thraustochytrium has been modified, that is to say functionally destroyed, for example by homologous recombination with a modified PSE gene or by mutagenesis and detection by means of DNA sequences. In a preferred embodiment, the plant organism belongs to the genus Physcomitrella, Ceratodon, Funaria, oilseed rape or linseed, with Physcomitrella, oilseed rape or linseed being preferred. In a preferred embodiment, Physcomitrella, oilseed rape or linseed is also used to produce a desired compound such as lipids or fatty acids, with PUFAs being especially preferred.
In yet another preferred embodiment, the moss Physcomitrella patens can be used for demonstrating a function of an elongase gene using homologous recombination on the basis of the nucleic acids described in the present invention.
Yet another aspect of the invention relates to an isolated PSE gene or a part, for example a biologically active part, thereof. In a preferred embodiment, the isolated PSE or a part thereof can participate in the metabolism of compounds required for the synthesis of cell membranes in a microorganism or a plant cell or in the transport of molecules via its membranes. In a further preferred embodiment, the isolated PSE or the part thereof has sufficient homology with an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12 for this protein or the part thereof to retain the ability to participate in the metabolism of compounds required for the synthesis of cell membranes in microorganisms or plant cells or in the transport of molecules via these membranes.
The invention also provides an isolated preparation of a PSE. In preferred embodiments, the PSE gene encompasses an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12. In a further preferred embodiment, the invention relates to an isolated full-length protein which is essentially homologous with a complete amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12 (which are encoded by the open reading frames shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO and SEQ ID NO:11). In a further embodiment, the protein has at least approximately 50%, preferably at least approximately 60%, more preferably at least approximately 70%, 80% or 90% and most preferably at least approximately 95%, 96%, 97%, 98%, 99% or more homology with an amino acid sequence of sequence SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12. In other embodiments, the isolated PSE encompasses an amino acid sequence which has at least approximately 50% homology with one of the amino acid sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12 and which can participate in the metabolism of compounds required for the synthesis of fatty acids in a microorganism or a plant cell or in the transport of molecules via these membranes or has one or more of the PUFA-elongating activities, the elongation advantageously concerning desaturated C16- or C18- or C20-carbon chains with double bonds in at least two positions.
As an alternative, the isolated PSE can encompass an amino acid sequence which is encoded by a nucleotide sequence hybridizing with a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11, for example under stringent conditions, or which has at least approximately 50%, preferably at least approximately 60%, more preferably at least approximately 70%, 80% or 90% and even more preferably at least approximately 95%, 96%, 97%, 98%, 99% or more homology thereto. It is also preferred for the preferred PSE forms also to have one of the PSE activities described herein.
The PSE polypeptide or a biologically active part thereof can be linked functionally to a non-PSE polypeptide to form a fusion protein. In preferred embodiments, this fusion protein has an activity which differs from that of PSE alone. In other preferred embodiments, this fusion protein participates in the metabolism of compounds which are required for the synthesis of lipids and fatty acids, cofactors and enzymes in microorganisms or plants, or in the transport of molecules via these membranes. In especially preferred embodiments, the introduction of this fusion protein into a host cell modulates the production of a desired compound by the cell. In a preferred embodiment, these fusion proteins also contain Δ4-, Δ5- or Δ6-, Δ8-, Δ15-, Δ17- or Δ19-desaturase activities, alone or in combination.
Another aspect of the invention relates to a process for the production of a fine chemical. This process either comprises culturing a suitable microorganism or culturing plant cells, plant tissues, plant organs or intact plants encompassing the nucleotide sequences according to the invention of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11 or their homologs, derivatives or analogs or a gene construct which compasses SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11 or their homologs, derivatives or analogs, or a vector encompassing these sequences or the gene construct which brings about the expression of a PSE nucleic acid molecule according to the invention so that a fine chemical is produced. In a preferred embodiment, the process furthermore encompasses the step of obtaining a cell comprising such an elongase nucleic acid sequence according to the invention, in which a cell is transformed with an elongase nucleic acid sequence, a gene construct or a vector which bring about the expression of a PSE nucleic acid according to the invention. In a further preferred embodiment, this process furthermore comprises the step of obtaining the fine chemical from the culture. In an especially preferred embodiment, the cell belongs to the order of the Ciliata, to microorganisms such as fungi, or to the plant kingdom, in particular to oil crops, with microorganisms or oil crops being especially preferred.
A further aspect of the invention relates to methods of modulating the production of a molecule by a microorganism. These methods encompass combining the cell with a substance which modulates the PSE activity or the expression of the PSE nucleic acid so that a cell-associated activity is modified relative to the same activity in the absence of the substance. In a preferred embodiment, a metabolic pathway, or two metabolic pathways, of the cell for lipids and fatty acids, cofactors and enzymes is, or are, modulated or the transport of compounds via these membranes is modulated so that the yield or the production rate of a desired fine chemical by this microorganism is improved. The substance which modulates the PSE activity can be a substance which stimulates the PSE activity or the expression of the PSE nucleic acid or which can be used as intermediate in fatty acid biosynthesis. Examples of substances which stimulate the PSE activity or the expression of PSE nucleic acids are, inter alia, small molecules, active PSEs and nucleic acids encoding PSEs which have been introduced into the cell. Examples of substances which inhibit the PSE activity or PSE expression are, inter alia, small molecules and/or antisense PSE nucleic acid molecules.
A further aspect of the invention relates to methods of modulating the yields of a desired compound from a cell, which encompass introducing, into a cell, a wild-type or mutant PSE gene which is either kept on a separate plasmid or integrated into the genome of the host cell. In the case of integration into the genome, integration can be random or take place by recombination in such a way that the native gene is replaced by the copy which is introduced, thus modulating the production of the desired compound by the cell, or by using a gene in trans, so that the gene is functionally linked to a functional expression unit comprising at least one sequence which ensures the expression of a gene and at least one sequence which ensures the polyadenylation of a functionally transcribed gene.
In a preferred embodiment, the yields are modified. In a further embodiment, the desired chemical is increased, it being possible to reduce undesired compounds which have a negative effect. In an especially preferred embodiment, the desired fine chemical is a lipid or fatty acid, a cofactor or an enzyme. In an especially preferred embodiment, this chemical is a polyunsaturated fatty acid. More preferably, it is selected from amongst arachidonic acid (=ARA), eicosapentaenoic acid (=EPA) or docosahexaenoic acid (=DHA).